The pursuit of approaches for treatment and prevention of cancer and infectious diseases represents an ongoing effort in the medical community. Recent efforts to combat cancer and infectious disease have included attempts to induce or enhance immune responses in subjects suffering from a type of cancer or an infectious disease. See, e.g. Srivastava et al. (1998) Immunity 8:657–665.
Ischemia/reperfusion injury is a significant source of morbidity and mortality in a number of clinical disorders, including myocardial infarction, cerebrovascular disease, and peripheral vascular disease. In addition, ischemia/reperfusion is relevant to the function of transplanted organs and to the recovery expedience following any cardiovascular surgery. See Fan et al. (1999) J Mol Med 77:577–596. Thus, the identification of cellular protective mechanisms against ischemia-induced damage is a central goal for therapy of, for example, heart attacks, strokes, and neurodegenerative diseases, as well as for improvement of recovery following surgery or transplantation.
The Hsp90 class of molecular chaperones are among the most abundant proteins in eukaryotic cells. Hsp90 family members are phylogenetically ubiquitous whereas the endoplasmic reticulum paralog of HSP90, GRP94 (gp96, ERp99, endoplasmin), is found only in higher plants and metazoans (Nicchitta (1998) Curr Opin Immunol 10:103–109). The Hsp90 family of proteins are known to be involved in directing the proper folding and trafficking of newly synthesized proteins and in conferring protection to the cell during conditions of heat shock, oxidative stress, nutrient stress, and other physiological stress scenarios (Toft (1998) Trends Endocrinol Metab 9:238–243; Pratt (1998) Proc Soc Exp Biol Med 217:420–434). Under such stress conditions, protein folding, protein oligomeric assembly, and protein stability can be profoundly disrupted. It is the function of the Hsp90 family of proteins, in concert with other molecular chaperones, to assist in preventing and reversing stress-induced inactivation of protein structure and function.
At a molecular level, HSP90 function in protein folding is known to require the activity of a series of co-chaperones and accessory molecules, including Hsp70, p48Hip, p60Hop, p23, and FKBP52 (Prodromou et al. (1999) EMBO J 18:754–762; Johnson et al. (1996) J Steroid Biochem Mol Biol 56:31–37; Chang et al. (1997) Mol Cell Biol 17:318–325; Duina et al. (1996) Science 274:1713–1715; Chen et al. (1996) Mol Endocrinol 10:682–693; Smith et al. (1993) J Biol Chem 268:18365–18371; Dittmar et al. (1998) J Biol Chem 273:7358–7366; Kosano et al. (1998) J Biol Chem 273:3273–3279). These co-chaperones and accessory molecules participate in both concerted and sequential interactions with HSP90 and thereby serve to regulate its chaperone activity (Buchner (1999) Trends Biochem Sci 24:136–141; Pratt et al. (1996) Exs 77:79–95; Pratt (1998) Proc Soc Exp Biol Med 217:420–434; Caplan (1999) Trends Cell Biol 9:262–268).
In addition to the contribution of co-chaperone proteins to the regulation of HSP90 function, recent crystallographic studies have identified an ATP/ADP binding pocket in the N-terminal domain of yeast and human HSP90, suggesting that HSP90 activity is regulated through cyclic ATP binding and hydrolysis, as has been established for the Hsp70 family of chaperones (Kassenbrock & Kelly (1989) EMBO J 8:1461–1467; Flynn et al. (1989) Science 245:385–390; Palleros et al. (1991) Proc Natl Acad Sci USA 88:519–523; Sriram et al. (1997) Structure 5:403–14; Prodromou et al. (1997) Cell 90:65–75; Obermann et al. (1998) J Cell Biol 143:901–910; Csermely & Kahn (1991) J Biol Chem 266:4943–4950; Csermely et al. (1993) J Biol Chem 268:1901–1907; Sullivan et al. (1997) J Biol Chem 272:8007–8012; Scheibel et al. (1997) J Biol Chem 272:18608–18613; Scheibel et al. (1998) Proc Natl Acad Sci USA 95:1495–1499; Panaretou et al. (1998) EMBO J 17:4829–4836; Caplan (1999) Trends Cell Biol 9:262–268; Grenert et al. (1999) J Biol Chem 274:17525–17533).
It has also been reported that HSP90 contains motifs bearing significant similarities to the Walker “A” and “B” sequences associated with ATP binding (Csermely & Kahn (1991) J Biol Chem 266:4943–4950; Jakob et al. (1996) J Biol Chem 271:10035–10041). Although these sequences are substantially different from the consensus sequences found among serine and tyrosine kinases, they are homologous to the ATP binding sequence seen in the Hsp70 family of proteins (Csermely & Kahn (1991) J Biol Chem 266:4943–4950). Consistent with sequence predictions, ATP binding, autophosphorylation activity, and ATPase activity have all been demonstrated for HSP90, though these findings are not without controversy (Csermely & Kahn (1991) J Biol Chem 266:4943–4950; Nadeau et al. (1993) J Biol Chem 268:1479–1487, Jakob et al. (1996) J Biol Chem 271:10035–10041; Grenert et al. (1999) J Biol Chem 274:17525–17533; Scheibel et al. (1997) J Biol Chem 272:18608–18613; Prodromou et al. (1997) Cell 90:65–75).
In part because of the very low affinity of HSP90 for ATP, a role for ATP in the regulation of HSP90 function remained under question until crystallographic resolution of the N-terminal domain of yeast and human HSP90 in association with bound adenosine nucleotides (Prodromou et al. (1997) Cell 90:65–75; Obermann et al. (1998) J Cell Biol 143:901–910). Aided by atomic scale structural insights, amino acid residues critical for ATP binding and hydrolysis were subsequently identified and analyzed (Prodromou et al. (1997) Cell 90:65–75; Panaretou et al. (1998) EMBO J 17:4829–4836; Obermann et al. (1998) J Cell Biol 143:901–910). Thus, in the human HSP90, aspartate 93 (D128 for GRP94; D79 for yeast HSP90) provides a direct hydrogen bond interaction with the N6 group of the purine moiety of the adenosine ring and is essential for ATP binding (Prodromou et al. (1997) Cell 90:65–75; Obermann et al. (1998) J Cell Biol 143:901–910). Glutamate 47 (E82 for GRP94; E33 for yeast HSP90) was postulated to play an important catalytic role in ATP hydrolysis, based both on its location relative to bound nucleotide and through comparison with the ATP binding domain of E. coli DNA gyrase B (Prodromou et al. (1997) Cell 90:65–75; Obermann et al. (1998) J Cell Biol 143:901–910). In subsequent mutagenesis studies of yeast HSP90, it was observed that the D79 mutant was deficient in ATP binding and that E47 mutants were deficient in ATP hydrolysis activity (Obermann et al. (1998) J Cell Biol 143:901–910; Panaretou et al. (1998) EMBO J 17:4829–4836). As further evidence for a function of these residues in HSP90 activity, yeast containing either mutant form of HSP90 were inviable (Obermann et al. (1998) J Cell Biol 143:901–910; Panaretou et al. (1998) EMBO J 17:4829–4836).
Progress in the development of Hsp90-based therapeutic and other applications has been impeded by a lack of characterization of ligand interactions of Hsp90 proteins, including GRP94. Despite the above-described characterization of ATP interaction with HSP90, evidence in support of intrinsic ATP binding and ATPase activities with respect to GRP94 is controversial and, as with HSP90, a clear consensus regarding the molecular basis of an adenosine nucleotide-mediated regulation of GRP94-substrate interactions has yet to emerge (Jakob et al. (1996) J Biol Chem 271:10035–10041; Wearsch & Nicchitta (1997) J Biol Chem 272:5152–5156; Li and Srivastava (1993) EMBO J 12:3143–3151; Csermely et al. (1995) J Biol Chem 270:6381–6388; Csermely et al. (1998) Pharmacol Ther 79:129–168).
What is needed, then, is characterization of ligand interactions at the ligand binding pocket of a HSP90 protein, in particular GRP94 and HSP90. To this end, the present invention discloses an isolated and purified GRP94 LBD polypeptide. The disclosure herein also provides screening methods pertaining to the biological activity of Hsp90 proteins. Thus, the present invention meets a long-standing need in the art for methods and compositions that contribute to the understanding, diagnosis and treatment of disorders related to Hsp90 protein function.